In an effort to understand the structural and functional organization of A-MuLV, integrated proviral genome was molecularly cloned in E. coli using bacteriophage vector LambdagtWESoLambdaB. A physical map of the proviral genome was generated and revealed that restriction enzymes HIND III, Bam HI, Cla I, Sal I, Pva I and Bst EII each cleaved at a unique site in the viral genome. Cloned A-MuLV proviral DNA was shown to transform NIH/3T3 cells. Moreover, such transformants contained the rescuable A-MuLV genome. In order to localize the region of A-MuLV required for transformation, the biological activity of the proviral genome following exposure to different restriction enzymes was tested. A-MuLV DNA cleaved with Sac I, Bgl I, Pvu I, Bgl II and PST I lost detectable biologic activity. In contrast, cleavage of A-MuLV DNA with Hind III, Bam HI, Sal I and Cla I did not impair transformation ability. Subgenomic fragments of A-MuLV cloned in plasmid vector pBR322 confirmed the above findings and helped to map the region needed for transforming function of A-MuLV.